1. The document summarizes various processes involved in the synthesis and transport of proteins within cells, including the synthesis of secretory and integral membrane proteins in the ER and their transport through the secretory pathway to their destinations. 2. Key details covered include the roles of signal sequences, SRP, and the translocon in targeting proteins to the ER, and the roles of vesicles, SNARE proteins, and Rabs in intracellular transport. 3. Mechanisms for transporting proteins and other molecules across membranes like diffusion, channels, carriers, and endocytosis/exocytosis are also summarized.

1. M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

2. Synthesis of secretory
proteins - review1. N-terminal signal sequence is
synthesized
2. Signal bound by SRP, complex
docks with SRP receptor on ER
membrane
3. Signal sequence binds to
translocon, internal channel opens,
inserted into translocon
4. Polypeptide elongates, signal sequence cleaved
5. ER chaperones prevent faulty folding, carbohydrates added to specific
residues
6. Ribosomes released, recycle
7. C-terminus of protein drawn into ER lumen, translocon gate shuts, protein
assumes final conformation

3. Synthesis of integral membrane
protein• Integral membrane
protein may, or may not
have N-terminal signal
sequence
• In absence of N-terminal
signal sequence, internal
signal sequence bound by
SRP
•Animation:ERimport.mov
• SRP-protein-ribosome complex docks with SRP receptor, C-terminal
portion of protein cotranslationally inserted into lumen of ER
•Mature protein transverses ER bilayer forming integral membrane protein
•NOTE: Orientation of protein within membrane dependent upon cluster of
charged residues adjacent to internal signal sequence
•In presence of N-terminal signal sequence, integral membrane protein
produced by stop-transfer signal that forms transmembrane domain

4. Secretory
Pathway
• Once a protein has entered exocytotic
pathway, in general, it never returns to
cytosol (notable exception is misfolded
proteins - retrograde transport for
degradation)
• In the absence of a sorting signal,
protein will follow constitutive secretory
pathway (i.e., directed to plasma
membrane) in transport vesicles
• Some proteins contain retention signals
(e.g., KDEL in C-terminus of some ER
proteins)

5. Secretory
Pathway
• In specialized cells, regulated secretory pathway leads to
packaging of product in secretory vesicles

6. Asymmetry of proteins and lipids
maintained during membrane
assembly
 Orientation of a protein
(asymmetry) is determined upon
entry into ER, does not change
during transit to other
membrane/organelle
 Fusion of a vesicle with the
plasma membrane preserves the
orientation of any integral
proteins embedded in the vesicle
bilayer
 Animation: Secretion.mov

7. Small GTPases Act as Molecular
Switches
GDP GTP
GTP GDP
“Inactive” “Active”
GEF
Pi
GAP
GTP exchange for bound GDP, facilitated by Guanine-nucleotide Exchange
Factors (GEFs), “activates” protein (usually resulting in conformational
change). Hydrolysis of GTP → GDP, accelerated by GTPase-Activating
Proteins (GAPs), “inactivates” complex.
ARF - vesicular transport
Ran - nuclear transport
Rab - regulated secretion,
endocytosis, intracellular
transport
Rho - formation of actin
cytoskeleton
Ras - growth and
differentiation signaling
pathways

8. Intracellular Transport
VesiclesStep 1: Coat assembly initiated
Step 2: ARF recruits coat proteins
Step 3: Vesicle budding
Step 4: Coat disassembly
Step 5: Vesicle targeting (v-
SNARE)
Step 6: General fusion machinery
assembles (NSF, SNAP)
Step 7: Vesicle fusion
Step 8: Retrograde transport
NOTE: Botulinum B toxin, one of most lethal toxins known (most serious cause
of food poisoning), is a protease that cleaves synaptobrevin (one v-SNARE
involved in fusion of synaptic vesicles) and inhibits release of acetylcholine at
neuromuscular junction. Possibly fatal, depending on dose taken.

9. Signal sequences target
proteins to their correct
destinations
• Signal sequences identified for cytosolic
proteins destined for nucleus,
mitochondria, peroxisomes
• Animation: Targeting.mov
• Nuclear import via nuclear pore
complex. Bidirectional transport,
accomodates large, complex structures
(e.g., ribosomes), nuclear localization
signal (NLS) not cleaved during
transport.
• Mitochondrial (mt) genome encodes 13 proteins, must import remainder.
Matrix proteins must pass through outer and inner mt membranes. Proteins
must be unfolded by chaperone proteins before translocation. Signal
sequence usually cleaved.
• Peroxisomes can import intact oligomers (e.g., tetrameric catalase).
Zellweger Syndrome - mutation in genes (peroxins) involved in peroxisome
biogenesis (or certain peroxisomal enzymes)

10. Major mechanisms used to transfer
material and information across
membranesCross-membrane movement of small molecules
Diffusion (passive and facilitated)
Active Transport
Cross-membrane movement of large molecules
Endocytosis
Exocytosis
Signal transmission across membranes
Cell surface receptors
1. Signal transduction (e.g., glucagon → cAMP)
2. Signal internalization (coupled with
endocytosis, e.g., LDL receptor)
Movement to intracellular receptors (steroid hormones;
a form of diffusion)
Intercellular contact and communication
Table 43-11

11. Passive Mechanisms Move Some
Small Molecules Across Membranes
 Passive transport down electrochemical gradients by
simple or facilitated diffusion
 passive diffusion (e.g., gases) limited by concentration
gradient across membrane, solubility of solute, thermal
agitation of that specific molecule
 Active transport, against gradient, requires energy

12. Ion Channels Selectively
Transport Charged Molecules
 Specific channels for Na+
, K+
, Ca2+
, and Cl-
have
been identified
 Channels are very selective, in most cases, to
only one type of ion
 Subset of K+
channels (“K+
leak channels”) open in
“resting” cell
 make plasma membrane more permeable to K+
than
other ions, maintains membrane potential

13. Activities of Ion Channels Can Be
Regulated
 Channels are “gated” - open transiently
 Ligand-gated channels - specific molecule binds receptor, open
channel (e.g., acetylcholine)
 Voltage-gated channels - open (or close) in response to changes
in membrane potential
 Ion channel activities are affected by certain drugs
 Mutations in genes encoding ion channels can cause
specific diseases (e.g., Cystic fibrosis - mutations in CFTR,
a Cl-
channel)

14. Net diffusion of substance
depends on:
 Its concentration gradient across membrane - solutes
move from high to low concentration
 Electrical potential across membrane - solutes move
toward solution with opposite charge (inside of cell
usually has negative charge)
 Permeability coefficient of substance
 Hydrostatic pressure gradient across membrane - ↑
pressure will ↑ rate and force of collision with membrane
 Temperature - ↑ temperature will ↑ particle motion and
frequency of collisions between particles and membrane

15. Types of transport systems
 Classified by direction of movement and whether
one or more unique molecules are moved
 Uniport system moves one type of molecule bidirectionally
 Cotransport systems transfer one solute dependent upon
simultaneous or sequential transfer of another solute
 Symport - moves solutes in same direction (e.g., Na+
-sugar
transporters or Na+
-amino acid transporters)
 Antiport - moves two molecules in opposite directions (e.g., Na+
in
and Ca2+
out)

16. Transport with carrier
proteins Facilitated diffusion and
active transport used to
transport molecules that
cannot pass freely through
lipid bilayer by themselves
 Both involve carrier
proteins; show specificity
for ions, sugars, and amino
acids; and resemble a
substrate-enzyme reaction
(but with no covalent
interaction)
 But, facilitated diffusion
can be bidirectional, while
active transport usually
unidirectional
 And, active transport
always against gradient,
requires energy
 Specific binding site for
solute
 Carrier is saturable (has
maximum rate of transport
-Vmax)
 There is a binding constant
(Km) for the solute, so the
whole system has a Km
 Structurally similar
competitive inhibitors
block transport

17. Facilitated
Diffusion Some solutes diffuse across membranes down
electrochemical gradients more rapidly than expected from
size, charge, and partition coefficients
 “Ping-Pong” mechanism explains facilitated diffusion
 Carrier protein exists in two principal conformations:
 “Pong” state - exposed to high [solute], solutes bind to specific
sites on carrier protein
 Conformational change exposes carrier to lower [solute] - “ping”
state
 Process is reversible, net flux depends on concentration gradient

18. Facilitated
Diffusion Rate of solute entry into cell determined by:
 Concentration gradient across the membrane
 Amount of carrier available (key control step)
 Rapidity of solute-carrier interaction
 Rapidity of conformational change (both loaded and
unloaded carrier)
 Hormones regulate by
changing number of
transporters available
 e.g., insulin increase glucose
transport in fat and muscle by
recruiting transporters from
intracellular reserve

19. Active Transport
 Transport away from thermodynamic equilibrium
 Energy is required (from hydrolysis of ATP, electron movement, or
light)
 Maintenance of electrochemical gradients in biologic systems
consumes ~30-40% of total energy expenditure of cell
 Cells, in general, maintain low intracellular [Na+
] and high
intracellular [K+
], with net negative electrical potential inside
 Gradients maintained by Na+
-K+
ATPase
 Ouabain or digitalis (cardiac glycosides used to
treat congestive heart failure) inhibits ATPase by
binding to extracellular domain. (Raises intracellular
[Na+
], Na+
/Ca2+
antiporter functions less efficiently
with lower [Na+
] gradient, thus fewer Ca2+
ions
exported, intracellular [Ca2+
] increases causing muscle to contract
more strongly.)

20. Glucose Transport - Several
Mechanisms In adipocytes and muscle, glucose enters by facilitated
diffusion
 In intestinal cells, glucose and Na+
bind to different sites
on glucose transporter (symport)
 Na+
enters cell down electrochemical
gradient and “drags” glucose with it
 To maintain steep Na+
gradient,
Na+
-glucose symport depends on low
intracellular [Na+
] maintained by
Na+
-K+
pump
 A uniport allows glucose accumulated in cell to move across
different membrane toward a new equilibrium

21. Endocyto
sis
 Process by which cells take up large molecules
 Source of nutritional elements (e.g., proteins,
polynucleotides)
 Mechanism for regulating content of certain
membrane components (e.g., hormone receptors)
 Most endocytotic vesicles fuse with lysosomes
 hydrolytic enzymes digest macromolecules (yields amino
acids, simple sugars, and nucleotides)
 Two general types of endocytosis
 Phagocytosis - specialized cells (e.g., macrophages) ingest
large particles (viruses, bacteria)

22. Endocyto
sis Pinocytosis - property of all cells
 Fluid-phase pinocytosis - nonselective uptake of a solute by small
vesicles
 loss of membrane replaced by exocytosis
 Absorptive pinocytosis - receptor-mediated
selective process
 permits selective concentration of ligands from
medium, limits uptake of fluid or soluble
unbound macromolecules
 vesicles derived from coated pits (clathrin)
 fate of receptor/ligand depends of particular receptor
 e.g., LDL receptor recycled, LDL processed in lysosomes
 EGF receptor degraded (receptor downregulation)
Fluid-phase Receptor-mediated
endocytosis endocytosis

23. Exocyto
sis
 Most cells release macromolecules to the exterior
 Signal for regulated exocytosis is often a hormone
 binds to cell-surface receptor, induces local and transient
change in [Ca2+
] that triggers exocytosis
 Molecules released by exocytosis fall into 3 categories
 Attach to cell surface and become peripheral proteins (e.g.,
antigens)
 Become part of extracellular matrix (e.g., collagen)
 Enter extracellular fluid and signal other cells (e.g., insulin)

24. Mutations Affecting Membrane
Proteins Cause Diseases
 Membrane proteins classified as: receptors,
transporters, ion channels, enzymes, and
structural components
 Member of each class often glycosylated
 mutations affecting this process may alter function